1. Field of the Invention
The present disclosure relates to an anti-tumor necrosis factor-alpha antibody (TNF-α), and in particular relates to a humanized monoclonal antibody which is capable of highly neutralizing tumor necrosis factor-alpha, and an amino acid sequence thereof.
2. Description of the Related Art
Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine produced primarily by cells of the immune system, including macrophage and monocyte cells. TNF-α is present as a homotrimeric protein in which each subunit is initially translated as a 26 kDa transmembrane precursor protein. After being cleavaged at a site proximal to the transmembrane domain of TNF-α by the TNF-α converting enzyme (TACE), a soluble trimeric form of TNF-α is released and exerts its activity by binding to two structurally distinct type I and type II tumor necrosis factor receptors (TNFRI and TNFRII) on effector cells.
The transmembrane form of TNF-α is also known for its unique biologic functions, such as cytotoxic activity and polyclonal B cell activation, in a cell-to-cell contact manner (Mitoma et al., 2008). TNF-α has been proved to have a certain effect on autoimmune processes and has become a key therapy target for many autoimmune diseases (Feldmann, 2001). So far, some anti-TNF-α agents, like etanercept, adalimumab and infliximab have been approved by the Food and Drug Administration (FDA) of America, and all have the capability to neutralize the soluble form of TNF-α effectively as a major pharmacological mechanism of action. However, the binding effects of these antagonists on the transmembrane form of TNF-α are different, which may cause different results for clinical diseases (Taylor, 2010). For instance, etanercept is not clinically effective for the pathogenesis of granulomatous diseases, in which the transmembrane form of TNF-α may play a critical role (2008, Mitoma). Therefore, whether anti-TNF-α agents are capable of binding to the transmembrane form of TNF-α, is a prerequisite for triggering antibody dependent cell mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), apoptotic and outside-to-inside signaling mechanisms.
A major impediment for using the murine monoclonal antibody in clinical practice is that it may elicit human anti-murine antibody (HAMA) responses in patients (Owens and Young, 1994; Sandhu, 1992; Schroff et al., 1985). Hence, to improve efficiency in clinical use, genetic engineering technology has been employed to replace the murine content with the amino acid residues of human counterparts, which reduces the possibility of inducing immunogenicity in patients.
An ideal for antibody humanization is that it should be capable of maintaining specificity and affinity toward an antigen and reduce immunogenicity as much as possible. So far, many approaches have been used for antibody humanization, such as chimeric antibodies, which consists of murine antigen-binding variable regions fused genetically to human antibody constant regions, is the earliest attempt to reduce immunogenicity (Morrison et al., 1984). However, chimeric antibodies would still generate undesirable anti-variable region responses (Bruggemann et al., 1989). CDR-gtafting is another approach involving the transfer of the complementarity determining regions (CDRs) from a rodent antibody to the Fv frameworks (FRs) of a human antibody (Verhoeyen et al., 1988). Unfortunately, the interface changes between CDRs and new FRs may largely disturb the binding to the antigen. The initial CDR-grafted antibodies tend to lose parental binding affinity, and therefore require additional work for back-mutation of several murine framework amino acids, which are regarded to be crucial for CDR loop conformations (Queen et al., 1989). Humanization via variable domain resurfacing is another approach that can maintain the specificity and binding affinity of a parental antibody, which can reduce the immunogenicity of antibodies through the replacement of surface exposed residues in the murine FRs with those usually found in human antibodies (Fontayne et al., 2006; Padlan, 1991; Roguska et al., 1994; Staelens et al., 2006; Zhang et al., 2005). Although current molecular-biology techniques render this approach more straightforward in practice, determining the critical residues exposed in solvent on the surface of antibody is still difficult, especially when requiring a reliable computer model of the antibody (Fontayne et al., 2006).